Project 1 - Meenhard Herlyn and Keiran Smalley Our long-term goal is to develop new therapies for melanoma. Five year survival rates for patients with disseminated disease have remained at approximately 15% for the last 30 years. Our overall hypothesis is that melanoma is a curable disease, provided the right pathways are targeted. The discovery in 2002 of specific activating mutations in the BRAF gene, the V600E mutation, has made this gene the first bona fide therapeutic target in melanoma. Lesions from patients with superficial spreading melanoma that lack BRAF mutations often harbor N-Ras mutations suggesting that activation of the MARK (mitogen-activated protein kinase) pathway may be a critical step in the oncogenic transformation of melanoma. However, the fact that BRAF mutations are also found in non-malignant nevi suggests that BRAF mutations alone are not sufficient for malignant transformation. Our working hypothesis is that BRAF is an essential component of any future disseminated melanoma therapy, but that other pathways independent of BRAF will also need to be targeted. We are focusing on the phospho inositide-3-kinase (PI3K) pathway due to its critical role in cell survival. We will use unique in vitro and in vivo models of human metastatic melanoma that provide an optimal environment for experimental melanoma therapy. Specifically, we propose to: 1. Target melanoma cells with mutant BRAF in two-dimensional (2D) conventional cell culture and three-dimensional (3D) organotypic models using small molecule inhibitors. We are testing the following three hypotheses: a. Targeting BRAF/MEK alone is not sufficient for melanoma cell apoptosis; b. Targeting BRAF/MEK in combination with inhibitors of PI3K/Akt will overcome cell survival to induce apoptosis; c. Amplifications in BRAF and CDK4/Cyclin D1 contribute to BRAF/MEK inhibitor resistance in sub-groups of melanomas. 2: Determine how the inhibition of GSK3|3 induces apoptosis of melanoma cells. We are testing the following two hypotheses: a. GSK3|3 inhibitors induce apoptosis in melanoma cells through direct pharmacological activation of p53; b. GSK3|3 inhibitors induce apoptosis through down-regulation of Mdm2. The proposed studies represent a close collaboration between all members of the Program Project on the development of novel inhibitors for BRAF, PI3K, and GSKSp.